A small anionic protein (6500 MW) from bone has been highly purified and characterized as a gamma-carboxyglutamic acid (GLA) containing calcium binding protein (2 Ca ions/molecule). Gla was first discovered in prothrombin and is formed in a posttranslational enzymatic vitamin K dependent carboxylation reaction requiring bicarbonate ion which is inhibited by the anticoagulants dicumarol and warfarin. In bone, de novo synthesis of Gla was established by organ culture methods using NaH 14 CO3, and the vitamin K dependent formation of Gla demonstrated (a) in vivo in normal, K deficient and dicumarol treated chicks by measuring the Gla content of various bones, and (b) in vitro, in bone microsome preparations where the carboxylase enzyme activity is located. The composition and amino acid sequence of the purified bone protein shows it to be unrelated to the Gla-containing plasma proteins. The protein in bone is named osteocalcin and its abundance in bone (1 molecule/1-2 tropocollagen molecules) is of great significance. In addition, Gla is shown to be present in ectopic calcifications (scleroderma, dermatomyositis, altheromatous plaque) and a Gla-protein of different origin from bone or prothrombin has been purified from calcium containing renal calculi. It was also found that free Gla acid is excreted in the urine in normal patients and at increased levels in renal stone patients, and patients with dermatomyositis. This project will specifically investigate: (1) the nature of the biosynthetic precursor of osteocalcin in bone microsome preparations; (2) the de novo synthesis of Gla at ectopic calcification sites by assaying for carboxylase enzyme activity; (3) the identification of the Gla containing proteins present in the soft tissue pathological calcification disorders: (4) the Gla content in the urine of patients with various metabolic bone disorders to evaluate Gla excretion as a potentially useful parameter; (5) the Gla content of various human normal and pathological bone specimens will be examined: it is hoped that changes in the Gla content due to excessive bone formation and/or resorption will be revealed.